Disease Treatment Drug Based on Mesenchymal-Stem-Cell Mobilization

ABSTRACT

The present inventors identified many nuclear proteins contained in the extract of skin tissue by mass spectrometry, randomly selected multiple partial amino acid sequences of the nuclear proteins, chemically synthesized peptides consisting of the partial amino acid sequences, and examined their activity of mobilizing mesenchymal stem cells. As a result, it was found that these multiple peptides show the activity of mobilizing mesenchymal stem cells into peripheral blood, even though their amino acid sequences are completely different from each other. The inventors also found that fragment peptides of the nuclear proteins have therapeutic effects on diseases characterized by inflammation and abnormalities of the immune system (e.g., inflammatory bowel disease and psoriasis). Based on these findings, a new regenerative medicine technology that can overcome the problems of cell transplantation therapy is provided.

TECHNICAL FIELD

The present application relates to compositions for mobilizingmesenchymal stem cells and agents for treating diseases based on themobilization of mesenchymal stem cells.

BACKGROUND ART

Mesenchymal stem cells contained in bone marrow fluid and the like havethe ability to differentiate into various tissues (pluripotency) such asbone, cartilage, fat, muscle, nerve, and epithelium. In recent years,attempts have been widely made to perform regenerative medicine (celltransplantation therapy) using bone marrow-derived mesenchymal stemcells proliferated by culture. However, collection of bone marrow bloodcontaining mesenchymal stem cells is done with an invasive techniquewhich inserts a thick needle into the iliac bone repeatedly, therebyplacing a large burden on the donor. In addition, mesenchymal stem cellsgradually lose their proliferative ability and pluripotency whensubcultured continuously in vitro. Furthermore, culturing mesenchymalstem cells based on high quality control that guarantees the safety ofin vivo transplantation requires special culture equipment such as CPC(cell processing center), so the current situation is that it can becarried out only at a limited number of universities and companies.

CITATION LIST Non-Patent Literature

-   [NPL 1] PNAS 2011 Apr. 19; 108 (16): 6609-14

PATENT LITERATURE

-   [PTL 1] WO2008/053892-   [PTL 2] WO2009/133939-   [PTL 3] WO2012/147470

SUMMARY OF INVENTION Technical Problem

An objective of the present application is to develop a new regenerativemedicine technology that can overcome the problems of celltransplantation therapy.

Solution to Problem

The present inventors identified a large number of nucleoproteinscontained in an extract of skin tissue by mass spectrometry, randomlyselected a plurality of partial amino acid sequences of the identifiednucleoproteins, and chemically synthesized peptides consisting of thepartial amino acid sequences. Then, their activity of mobilizingmesenchymal stem cells was examined. As a result, it was found thatthese peptides show an activity of mobilizing mesenchymal stem cellsinto peripheral blood, even though their amino acid sequences arecompletely different from each other. It was also found that fragmentpeptides of the nucleoproteins have therapeutic effects on diseasescharacterized by inflammation and abnormalities of the immune system(e.g., inflammatory bowel disease and psoriasis). Based on thesefindings, a new regenerative medicine technology to overcome theproblems of cell transplantation therapy is provided.

Specifically, the present application provides the following:[1]

A composition for use in mobilizing mesenchymal stem cells to peripheralblood, which comprises a nuclear protein or a fragment peptide thereof.

[2]

A composition for use in treatment of a disease or pathologicalcondition in a subject by mobilizing mesenchymal stem cells toperipheral blood, which comprises a nuclear protein or a fragmentpeptide thereof.

[3]

The composition of [2], wherein the treatment of a disease orpathological condition is selected from inflammation-suppressingtherapy, immunomodulatory therapy, tissue regeneration-inducing therapy,and tissue fibrosis-suppressing therapy.

[4]

The composition of [2], wherein the disease or pathological condition isselected from an inflammatory disease, an autoimmune disease, a diseaseaccompanied by tissue damage, ischemia, or necrosis, and a fibroticdisease.

[5]

The composition of [2], wherein the disease or pathological condition isselected from inflammatory bowel disease and psoriasis.

[6]

A composition for use in treatment of a disease selected frominflammatory bowel disease and psoriasis, which comprises a nuclearprotein or a fragment peptide thereof.

[7]

The composition of any one of [1] to [6], wherein the nuclear protein orfragment peptide thereof comprises a nuclear localization signal.

[8]

The composition of any one of [1] to [7], wherein the nuclear protein orfragment peptide thereof is a nuclear protein involved in transcriptionregulation or a fragment peptide thereof.

[9]

The composition of any one of [1] to [8], wherein the nuclear protein orfragment peptide thereof is a nuclear protein selected from thefollowing or a fragment peptide thereof:

(1) BTF3 protein;(2) SUPT16H protein;(3) YBX1 protein;(4) NPM1 protein;(5) PA2G4 protein;(6) PFDN5 protein;(7) PSMC3 protein;(8) HNRNPK protein; and(9) a nuclear protein functionally equivalent to a protein selected from(1) to (8).[10]

The composition of any one of [1] to [9], wherein the nuclear protein orfragment peptide thereof is a nuclear protein selected from thefollowing or a fragment peptide thereof:

(a) a nuclear protein comprising an amino acid sequence selected fromSEQ ID NOs: 1 to 34;(b) a nuclear protein comprising an amino acid sequence resulting fromsubstitution, deletion, insertion, or addition of one or more aminoacids in an amino acid sequence selected from SEQ ID NOs: 1 to 34; and(c) a nuclear protein comprising an amino acid sequence with a sequenceidentity of about 80% or higher with an amino acid sequence selectedfrom SEQ ID NOs: 1 to 34.[11]

The composition of any one of [1] to [10], wherein the fragment peptideof the nuclear protein is a fragment peptide selected from thefollowing:

(a) a nuclear protein fragment peptide consisting of a portion of anamino acid sequence selected from SEQ ID NOs: 1 to 34;(b) a nuclear protein fragment peptide comprising an amino acid sequenceselected from SEQ ID NOs: 35 to 56;(c) a nuclear protein fragment peptide consisting of a portion of anamino acid sequence selected from SEQ ID NOs: 35 to 56;(d) a nuclear protein fragment peptide comprising an amino acid sequenceresulting from substitution, deletion, insertion, or addition of one ormore amino acids in an amino acid sequence selected from SEQ ID NOs: 35to 56; and(e) a nuclear protein fragment peptide comprising an amino acid sequencewith a sequence identity of about 80% or higher with an amino acidsequence selected from SEQ ID NOs: 35 to 56.[12]

A fragment peptide of a nuclear protein selected from the following:

(1) BTF3 protein;(2) SUPT16H protein;(3) YBX1 protein;(4) NPM1 protein;(5) PA2G4 protein;(6) PFDN5 protein;(7) PSMC3 protein;(8) HNRNPK protein; and(9) a nuclear protein functionally equivalent to a protein selected from(1) to (8).[13]

The fragment peptide of [12], comprising a nuclear localization signal.

[14]

The fragment peptide of [12] or [13], which is a fragment peptide of anuclear protein selected from the following:

(a) a nuclear protein comprising an amino acid sequence selected fromSEQ ID NOs: 1 to 34;(b) a nuclear protein comprising an amino acid sequence resulting fromsubstitution, deletion, insertion, or addition of one or more aminoacids in an amino acid sequence selected from SEQ ID NOs: 1 to 34; and(c) a nuclear protein comprising an amino acid sequence with a sequenceidentity of about 80% or higher with an amino acid sequence selectedfrom SEQ ID NOs: 1 to 34.[15]

The fragment peptide of any one of [12] to [14], which is selected fromthe following:

(a) a nuclear protein fragment peptide consisting of a portion of anamino acid sequence selected from SEQ ID NOs: 1 to 34;(b) a nuclear protein fragment peptide comprising an amino acid sequenceselected from SEQ ID NOs: 35 to 56;(c) a nuclear protein fragment peptide consisting of a portion of anamino acid sequence selected from SEQ ID NOs: 35 to 56;(d) a nuclear protein fragment peptide comprising an amino acid sequenceresulting from substitution, deletion, insertion, or addition of one ormore amino acids in an amino acid sequence selected from SEQ ID NOs: 35to 56; and(e) a nuclear protein fragment peptide comprising an amino acid sequencewith a sequence identity of about 80% or higher with an amino acidsequence selected from SEQ ID NOs: 35 to 56.

[A1]

A method for mobilizing mesenchymal stem cells to peripheral blood,comprising administering to a subject an effective amount of a nuclearprotein or a fragment peptide thereof.

[A2]

A method for treating a disease or pathological condition in a subjectby mobilizing mesenchymal stem cells to peripheral blood, whichcomprises administering to the subject an effective amount of a nuclearprotein or a fragment peptide thereof.

[A3]

The method of [A2], wherein the treatment of a disease or pathologicalcondition is selected from inflammation-suppressing therapy,immunomodulatory therapy, tissue regeneration-inducing therapy, andtissue fibrosis-suppressing therapy.

[A4]

The method of [A2], wherein the disease or pathological condition isselected from an inflammatory disease, an autoimmune disease, a diseaseaccompanied by tissue damage, ischemia, or necrosis, and a fibroticdisease.

[A5]

The method of [A2], wherein the disease or pathological condition isselected from inflammatory bowel disease and psoriasis.

[A6]

A method for treating a disease selected from inflammatory bowel diseaseor psoriasis in a subject, which comprises administering to the subjectan effective amount of a nuclear protein or a fragment peptide thereof.

[A7]

The method of any one of [A1] to [A6], wherein the nuclear protein orfragment peptide thereof comprises a nuclear localization signal.

[A8]

The method of any one of [A1] to [A7], wherein the nuclear protein orfragment peptide thereof is a nuclear protein involved in transcriptionregulation or a fragment peptide thereof.

[A9]

The method of any one of [A1] to [A8], wherein the nuclear protein orfragment peptide thereof is a nuclear protein selected from thefollowing or a fragment peptide thereof:

(1) BTF3 protein;(2) SUPT16H protein;(3) YBX1 protein;(4) NPM1 protein;(5) PA2G4 protein;(6) PFDN5 protein;(7) PSMC3 protein;(8) HNRNPK protein; and(9) a nuclear protein functionally equivalent to a protein selected from(1) to (8).

[A10]

The method of any one of [A1] to [A9], wherein the nuclear protein orfragment peptide thereof is a nuclear protein selected from thefollowing or a fragment peptide thereof:

(a) a nuclear protein comprising an amino acid sequence selected fromSEQ ID NOs: 1 to 34;(b) a nuclear protein comprising an amino acid sequence resulting fromsubstitution, deletion, insertion, or addition of one or more aminoacids in an amino acid sequence selected from SEQ ID NOs: 1 to 34; and(c) a nuclear protein comprising an amino acid sequence with a sequenceidentity of about 80% or higher with an amino acid sequence selectedfrom SEQ ID NOs: 1 to 34.

[A11]

The method of any one of [A1] to [A10], wherein the fragment peptide ofthe nuclear protein is a fragment peptide selected from the following:

(a) a nuclear protein fragment peptide consisting of a portion of anamino acid sequence selected from SEQ ID NOs: 1 to 34;(b) a nuclear protein fragment peptide comprising an amino acid sequenceselected from SEQ ID NOs: 35 to 56;(c) a nuclear protein fragment peptide consisting of a portion of anamino acid sequence selected from SEQ ID NOs: 35 to 56;(d) a nuclear protein fragment peptide comprising an amino acid sequenceresulting from substitution, deletion, insertion, or addition of one ormore amino acids in an amino acid sequence selected from SEQ ID NOs: 35to 56; and(e) a nuclear protein fragment peptide comprising an amino acid sequencewith a sequence identity of about 80% or higher with an amino acidsequence selected from SEQ ID NOs: 35 to 56.

[B1]

A nuclear protein or a fragment peptide thereof for use in mobilizingmesenchymal stem cells to peripheral blood.

[B2]

A nuclear protein or a fragment peptide thereof for use in treatment ofa disease or pathological condition in a subject by mobilizingmesenchymal stem cells to peripheral blood.

[B3]

The nuclear protein or fragment peptide thereof of [B2], wherein thetreatment of a disease or pathological condition is selected frominflammation-suppressing therapy, immunomodulatory therapy, tissueregeneration-inducing therapy, and tissue fibrosis-suppressing therapy.

[B4]

The nuclear protein or fragment peptide thereof of [B2], wherein thedisease or pathological condition is selected from an inflammatorydisease, an autoimmune disease, a disease accompanied by tissue damage,ischemia, or necrosis, and a fibrotic disease.

[B5]

The nuclear protein or fragment peptide thereof of [B2], wherein thedisease or pathological condition is selected from inflammatory boweldisease and psoriasis.

[B6]

A nuclear protein or a fragment peptide thereof for use in treatment ofa disease selected from inflammatory bowel disease and psoriasis.

[B7]

The nuclear protein or fragment peptide thereof of any one of [B1] to[B6], which comprises a nuclear localization signal.

[B8]

The nuclear protein or fragment peptide thereof of any one of [B1] to[B7], which is a nuclear protein involved in transcription regulation ora fragment peptide thereof.

[B9]

The nuclear protein or fragment peptide thereof of any one of [B1] to[B8], which is selected from the following:

(1) BTF3 protein;(2) SUPT16H protein;(3) YBX1 protein;(4) NPM1 protein;(5) PA2G4 protein;(6) PFDN5 protein;(7) PSMC3 protein;(8) HNRNPK protein; and(9) a nuclear protein functionally equivalent to a protein selected from(1) to (8).

[B10]

The nuclear protein or fragment peptide thereof of any one of [B1] to[B9], which is selected from the following:

(a) a nuclear protein comprising an amino acid sequence selected fromSEQ ID NOs: 1 to 34;(b) a nuclear protein comprising an amino acid sequence resulting fromsubstitution, deletion, insertion, or addition of one or more aminoacids in an amino acid sequence selected from SEQ ID NOs: 1 to 34; and(c) a nuclear protein comprising an amino acid sequence with a sequenceidentity of about 80% or higher with an amino acid sequence selectedfrom SEQ ID NOs: 1 to 34.

[B11]

The nuclear protein or fragment peptide thereof of any one of [B1] to[B10], which is selected from the following:

(a) a nuclear protein fragment peptide consisting of a portion of anamino acid sequence selected from SEQ ID NOs: 1 to 34;(b) a nuclear protein fragment peptide comprising an amino acid sequenceselected from SEQ ID NOs: 35 to 56;(c) a nuclear protein fragment peptide consisting of a portion of anamino acid sequence selected from SEQ ID NOs: 35 to 56;(d) a nuclear protein fragment peptide comprising an amino acid sequenceresulting from substitution, deletion, insertion, or addition of one ormore amino acids in an amino acid sequence selected from SEQ ID NOs: 35to 56; and(e) a nuclear protein fragment peptide comprising an amino acid sequencewith a sequence identity of about 80% or higher with an amino acidsequence selected from SEQ ID NOs: 35 to 56.

[C1]

Use of a nuclear protein or a fragment peptide thereof in manufacture ofa medicament or reagent for mobilizing mesenchymal stem cells toperipheral blood.

[C2]

Use of a nuclear protein or a fragment peptide thereof in manufacture ofa medicament for treating a disease or pathological condition in asubject by mobilizing mesenchymal stem cells to peripheral blood.

[C3]

The use of [C2], wherein the treatment of a disease or pathologicalcondition is selected from inflammation-suppressing therapy,immunomodulatory therapy, tissue regeneration-inducing therapy, andtissue fibrosis-suppressing therapy.

[C4]

The use of [C2], wherein the disease or pathological condition isselected from an inflammatory disease, an autoimmune disease, a diseaseaccompanied by tissue damage, ischemia, or necrosis, and a fibroticdisease.

[C5]

The use of [C2], wherein the disease or pathological condition isselected from inflammatory bowel disease and psoriasis.

[C6]

Use of a nuclear protein or a fragment peptide thereof in manufacture ofa medicament for treating a disease selected from inflammatory boweldisease and psoriasis.

[C7]

The use of any one of [C1] to [C6], wherein the nuclear protein orfragment peptide thereof comprises a nuclear localization signal.

[C8]

The use of any one of [C1] to [C7], wherein the nuclear protein orfragment peptide thereof is a nuclear protein involved in transcriptionregulation or a fragment peptide thereof.

[C9]

The use of any one of [C1] to [C8], wherein the nuclear protein orfragment peptide thereof is a nuclear protein selected from thefollowing or a fragment peptide thereof:

(1) BTF3 protein;(2) SUPT16H protein;(3) YBX1 protein;(4) NPM1 protein;(5) PA2G4 protein;(6) PFDN5 protein;(7) PSMC3 protein;(8) HNRNPK protein; and(9) a nuclear protein functionally equivalent to a protein selected from(1) to (8).

[C10]

The use of any one of [C1] to [C9], wherein the nuclear protein orfragment peptide thereof is a nuclear protein selected from thefollowing or a fragment peptide thereof:

(a) a nuclear protein comprising an amino acid sequence selected fromSEQ ID NOs: 1 to 34;(b) a nuclear protein comprising an amino acid sequence resulting fromsubstitution, deletion, insertion, or addition of one or more aminoacids in an amino acid sequence selected from SEQ ID NOs: 1 to 34; and(c) a nuclear protein comprising an amino acid sequence with a sequenceidentity of about 80% or higher with an amino acid sequence selectedfrom SEQ ID NOs: 1 to 34.

[C11]

The use of any one of [C1] to [C10], wherein the fragment peptide of thenuclear protein is a fragment peptide selected from the following:

(a) a nuclear protein fragment peptide consisting of a portion of anamino acid sequence selected from SEQ ID NOs: 1 to 34;(b) a nuclear protein fragment peptide comprising an amino acid sequenceselected from SEQ ID NOs: 35 to 56;(c) a nuclear protein fragment peptide consisting of a portion of anamino acid sequence selected from SEQ ID NOs: 35 to 56;(d) a nuclear protein fragment peptide comprising an amino acid sequenceresulting from substitution, deletion, insertion, or addition of one ormore amino acids in an amino acid sequence selected from SEQ ID NOs: 35to 56; and(e) a nuclear protein fragment peptide comprising an amino acid sequencewith a sequence identity of about 80% or higher with an amino acidsequence selected from SEQ ID NOs: 35 to 56.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a plot of the number of colonies obtained by culturingperipheral blood 14 hours after administration of saline or thepeptides. The number of colonies is shown as a value converted per 1 mLof collected peripheral blood. The long horizontal bar represents themean value, and the short horizontal bars represent the standarddeviation.

FIG. 2 is a plot of the number of colonies obtained by culturingperipheral blood 14 hours after administration of saline or thepeptides. The number of colonies is shown as a value converted per 1 mLof collected peripheral blood. The long horizontal bar represents themean value, and the short horizontal bars represent the standarddeviation.

FIG. 3 is a plot of the number of colonies obtained by culturingperipheral blood 16 hours after administration of saline or thepeptides. The number of colonies is shown as a value per peripheralblood volume (about 800 μL) collected from one mouse. The longhorizontal bar represents the average value, and the short horizontalbars represent the standard deviation.

FIG. 4 is a plot of the number of colonies obtained by culturingperipheral blood 24 hours after administration of saline or thepeptides. The number of colonies is shown as a value per peripheralblood volume (about 800 μL) collected from one mouse. The longhorizontal bar represents the average value, and the short horizontalbars represent the standard deviation.

FIG. 5 is a graph that shows the body weight change of the mice. In thegraph, “saline” indicates the control group, and “NP-1” indicates thepeptide NP-1 administration group. On the horizontal axis, the number ofdays indicates the number of days after the start of drinking theaqueous solution of dextran sulfate sodium (DSS), and triangle indicatesthe date of administration of physiological saline (control group) orthe peptide (NP-1 administration group).

FIG. 6 is a graph that shows the body weight change of the mice. In thegraph, “saline” indicates the control group, and “NP-2” indicates thepeptide NP-2 administration group. On the horizontal axis, the number ofdays indicates the number of days after the start of drinking theaqueous solution of dextran sulfate sodium (DSS), and triangle indicatesthe date of administration of physiological saline (control group) orthe peptide (NP-2 administration group).

FIG. 7 is a graph that shows the body weight change of the mice. In thegraph, “saline” indicates the control group, and “NP-3” indicates thepeptide NP-3 administration group. On the horizontal axis, the number ofdays indicates the number of days after the start of drinking theaqueous solution of dextran sulfate sodium (DSS), and triangle indicatesthe date of administration of physiological saline (control group) orthe peptide (NP-3 administration group).

FIG. 8 is a graph that shows the body weight change of the mice. In thegraph, “saline” indicates the control group, and “NP-4” indicates thepeptide NP-4 administration group. On the horizontal axis, the number ofdays indicates the number of days after the start of drinking theaqueous solution of dextran sulfate sodium (DSS), and triangle indicatesthe date of administration of physiological saline (control group) orthe peptide (NP-4 administration group).

FIG. 9 is a graph showing changes in auricular thickness of mice. “Nontreat” indicates normal mice, “IMQ/saline” indicates the control group,and “IMQ/NP-3” indicates the peptide NP-3 administration group. Thehorizontal axis indicates the number of days after the start ofimiquimod application, and the vertical axis (Δthickness) showsdifference (An−A0) between the auricular thickness before the start ofimiquimod application (Day 0) (A0) and the auricular thickness at eachday after the start of imiquimod application (An; n=1-7).

DESCRIPTION OF EMBODIMENTS

The present inventors previously found a substance having an activity ofactivating stem cells in a living body or mobilizing them to an injuredtissue via peripheral circulation, and believe that the substance ispromising as a new type of medicine that can overcome the weaknesses ofcell therapy. Specifically, the present inventors have found that HighMobility Group Box 1 (HMGB1), which is a nuclear protein released fromnecrotic tissue, mobilizes cells that are positive for platelet-derivedgrowth factor receptor α (PDGFRα), which play a role in inducing tissueregeneration in vivo (believed to be mesenchymal stem cells (MSC)), to anecrotic tissue through peripheral circulation, thereby suppressesinflammation of the necrotic tissue and promotes tissue regeneration.Further, the present inventors have also found that fragment peptides ofHMGB1 exhibit an activity of mobilizing mesenchymal stem cells intoperipheral blood and a tissue regeneration-inducing activity. Therefore,the activities shown by the HMGB1 fragment peptides do not appear todepend on three-dimensional structure.

HMGB1 is physiologically absent in the blood and is released into theblood by necrotic cells only when necrotic damage occurs. This suggeststhat mesenchymal stem cells recognize the presence of necrotic tissue invivo by the exposure to HMGB1 or a fragment thereof, and are thenmobilized into peripheral blood to suppress inflammation of the necrotictissue and promote regeneration.

Here, the present inventors considered that nuclear proteins other thanHMGB1 would also be released into the blood by necrotic damage proteins,and that nuclear proteins other than HMGB1 or fragments thereof may havesimilar mesenchymal stem cell-mobilizing activity as the HMGB1 proteinor fragment peptides thereof. That is, the inventors considered that itwould be possible to provide the theory that “mesenchymal stem cellsrecognize nuclear proteins or fragment peptides thereof that are notphysiologically present in the blood and are mobilized to the blood”.

To prove this theory, the present inventors identified many nuclearproteins contained in skin tissue extracts by mass spectrometry,randomly selected multiple partial amino acid sequences of the nuclearproteins identified, chemically synthesized peptides consisting of thepartial amino acid sequences, and examined them for the mesenchymal stemcell-mobilizing activity. As a result, the above theory was confirmed tobe correct, as these multiple peptides showed activities to mobilizemesenchymal stem cells into peripheral blood even though their aminoacid sequences were completely different from each other.

Further, the present inventors found that fragment peptides of theabove-mentioned nuclear proteins show therapeutic effects againstdiseases characterized by inflammation and abnormalities of the immunesystem (such as inflammatory bowel disease and psoriasis). Specifically,it was confirmed that the fragment peptides of nuclear proteins suppressweight loss in a mouse model of inflammatory bowel disease and suppressskin thickening in a psoriasis model.

It is well known to those skilled in the art that mesenchymal stem cellsexert anti-inflammatory, immunomodulatory, and antifibrotic effects.Further, it is also well known to those skilled in the art thatmesenchymal stem cells can exert a regeneration-promoting action ondamaged tissues, as they have multipotency in that they candifferentiate into various tissues. Therefore, by administering to asubject a nuclear protein or a fragment peptide thereof that has anactivity of mobilizing mesenchymal stem cells to peripheral blood, themesenchymal stem cells are recruited into the peripheral blood, andtherapeutic effects for various diseases may be provided by themesenchymal stem cells' anti-inflammatory action, immunomodulatoryaction, anti-fibrotic action, and tissue regeneration-promoting action(due to the differentiation and/or anti-inflammatory action ofmesenchymal stem cells).

The present application provides compositions containing a nuclearprotein or a fragment peptide thereof for use in mobilizing mesenchymalstem cells into peripheral blood.

The compositions for use in mobilizing mesenchymal stem cells into theperipheral blood of the present application can be used as apharmaceutical composition or a reagent composition. In the presentapplication, the term “pharmaceutical composition” is usedinterchangeably with “medicament”, “drug” or “pharmacologicalcomposition”, and the term “reagent composition” is used interchangeablywith “reagent”.

The compositions for use in mobilizing mesenchymal stem cells intoperipheral blood of the present application can be used for treating adisease or pathological condition in a subject, for example, bymobilizing mesenchymal stem cells into peripheral blood.

Mesenchymal stem cells mobilized into peripheral blood using thecomposition for mobilizing mesenchymal stem cells into the peripheralblood of the present application can also be collected from the body,concentrated, and then used for treatment of a disease or pathologicalcondition in a subject. The present application also provides use of anuclear protein or a fragment thereof in the manufacture of a medicamentor a reagent for collecting mesenchymal stem cells from the body.

The compositions for use in mobilizing mesenchymal stem cells into theperipheral blood of the present application can also be used in, forexample, basic research, clinical research and such. Basic research andclinical research include, but are not limited to, mesenchymal stem cellmobilization research in vitro and mesenchymal stem cell mobilizationresearch in laboratory animals. The present application also providesuse of nuclear proteins or fragment peptides thereof in the manufactureof pharmaceuticals or reagents for basic or clinical research.

The compositions for use in mobilizing mesenchymal stem cells intoperipheral blood can comprise one or more nuclear proteins, one or morefragment peptides, or a combination thereof.

In the present application, “mesenchymal stem cells” are cells that arecollected from bone marrow or other tissues (blood such as umbilicalcord blood, and skin, fat, pulp, etc.), can be cultured and proliferatedon culture dishes (plastic or glass) as adherent cells, and have theability to differentiate into mesenchymal tissues such as bone,cartilage, fat, and muscle. In one embodiment, mesenchymal stem cellsalso have the ability to differentiate into epithelial tissues and nervetissues. In one embodiment, mesenchymal stem cells are cells capable offorming colonies. In the present application, mesenchymal stem cells mayexist as a heterogeneous cell population comprising not only stem cellsin the narrow sense (cells having self-renewal ability anddifferentiation ability) but also progenitor cells. Under cultureconditions, the mesenchymal stem cells may include stem cells in thenarrow sense, or may even include differentiated cells in addition tostem cells in the narrow sense and progenitor cells. In one embodiment,the mesenchymal stem cells may be composed only of stem cells in thenarrow sense.

In the present application, progenitor cells are defined as cells with aunidirectional ability to differentiate into cells of specific tissuesother than the blood system, and include cells that have the ability todifferentiate into mesenchymal tissues, epithelial tissues, nervetissues, parenchymatous organs, vascular endothelium.

In the present application, the mesenchymal stem cells include, but arenot limited to, bone marrow mesenchymal stem cells and bonemarrow-derived mesenchymal stem cells. The “bone marrow mesenchymal stemcells” exist in the bone marrow, and may be harvested from bone marrowand cultured and proliferated as adherent cells on culture dish (made ofplastic or glass); and they are cells characterized in having theability to differentiate into mesenchymal tissues such as bone,cartilage, fat, muscle and such. In one embodiment, bone marrowmesenchymal stem cells also have the ability to differentiate intoepithelial tissues and nerve tissues. In one embodiment, bone marrowmesenchymal stem cells are cells capable of forming colonies. In thepresent application, the term “bone marrow mesenchymal stem cell” isused interchangeably with “bone marrow mesenchymal stromal cell”, “bonemarrow pluripotent stem cell” or “bone marrow pluripotent stromal cell”.

“Bone marrow-derived mesenchymal stem cells” refers to bone marrowmesenchymal stem cells that have been mobilized from bone marrow to theoutside of the bone marrow, and are cells that can be collected byperipheral blood collection, and further from mesenchymal tissues suchas fat, epithelial tissues such as skin, or nerve tissues such as thebrain. In the present application, the term “bone marrow-derivedmesenchymal stem cell” can be used interchangeably with “bonemarrow-derived mesenchymal stromal cell”, “bone marrow-derivedpluripotent stem cell” or “bone marrow-derived pluripotent stromalcell”.

In one embodiment, bone marrow mesenchymal stem cells and bonemarrow-derived mesenchymal stem cells are also characterized in that, bybeing administered to an injured part of a living body directly aftercollection or after once attached to a culture dish, the cells are alsocapable of differentiating into, for example, epithelial tissues such asskin-constituting keratinocytes or tissues of the nerve system whichconstitutes the brain.

Bone marrow mesenchymal stem cells and bone marrow-derived mesenchymalstem cells preferably have the ability to differentiate into osteoblastcells (identifiable by calcium deposition observed when differentiationis induced), cartilage cells (identifiable by being Alcian bluestaining-positive, safranin-O staining-positive, or such), and fat cells(identifiable by being Sudan III staining-positive or such), and alsodifferentiate into, for example, mesenchymal cells such as fibroblasts,smooth muscle cells, skeletal muscle cells, stromal cells, and tendoncells, nerve cells, pigment cells, epidermal cells, hair follicle cells(expressing cytokeratin family, hair keratin family or such), epithelialcells (for example, epithelial keratinized cells and intestinalepithelial cells express cytokeratin family or such), endothelial cells,and further differentiate into cells of parenchymal organs such asliver, kidney and pancreas, but the differentiated cells are not limitedto the above cells.

Human mesenchymal stem cell markers can be exemplified by some or all ofPDGFRα positive, PDGFRβ positive, Lin negative, CD45 negative, CD44positive, CD90 positive, CD29 positive, Flk-1 negative, CD105 positive,CD73 positive, CD90 positive, CD71 positive, Stro-1 positive, CD106positive, CD166 positive, CD31 negative, CD271 positive, and CD11bnegative, but are not limited thereto.

Murine mesenchymal stem cell markers can be exemplified by some or allof CD44 positive, PDGFRα positive, PDGFRβ positive, CD45 negative, Linnegative, Sea-1 positive, c-kit negative, CD90 positive, CD105 positive,CD29 positive, Flk-1 negative, CD271 positive, and CD11b negative, butare not limited thereto.

Rat mesenchymal stem cell markers can be exemplified by some or all ofPDGFRα positive, CD44 positive, CD54 positive, CD73 positive, CD90positive, CD105 positive, CD29 positive, CD271 positive, CD31 negative,and CD45 negative, but are not limited thereto.

In the present application, examples of mesenchymal stem cells includePDGFRα-positive mesenchymal stem cells, PDGFRα-positive bonemarrow-derived mesenchymal stem cells, and PDGFRα-positive bonemarrow-derived cells obtained as adherent cells by cell culture of amononuclear cell fraction in blood obtained by bone marrow harvest (bonemarrow cell collection) or peripheral blood collection, but they are notlimited thereto. Examples of PDGFRα-positive mesenchymal stem cellsinclude PDGFRα- and CD44-positive cells, PDGFRα- and CD90-positivecells, PDGFRα- and CD105-positive cells, PDGFRα- and CD29-positivecells, and such. In one embodiment, PDGFRα-positive mesenchymal stemcells may be CD44-negative cells.

The present application provides compositions for use in the treatmentof a disease or a pathological condition in a subject by mobilizingmesenchymal stem cells into the peripheral blood, comprising a nuclearprotein or a fragment peptide thereof.

The compositions for use in the treatment of a disease or a pathologicalcondition in a subject by mobilizing mesenchymal stem cells into theperipheral blood in the present application can be used aspharmaceutical compositions.

The subject in the present application is not particularly limited, andexamples thereof include mammals, birds, and fish. Mammals include humanand non-human animals, for example, human, mouse, rat, monkey, pig, dog,rabbit, hamster, guinea pig, horse, sheep, and whale, but are notlimited thereto. In the present application, the term “subject” is usedinterchangeably with “patient”, “individual”, or “animal”.

The composition for use in the treatment of a disease or pathologicalcondition in a subject by mobilization of mesenchymal stem cells intothe peripheral blood in the present application can comprise one or morenuclear proteins, one or more fragment peptides, or combinationsthereof.

In the present application, the treatment of a disease or pathologicalcondition is selected from, for example, inflammation-suppressingtherapy, immunomodulatory therapy, tissue regeneration-inducing therapy,and tissue fibrosis-suppressing therapy, but is not limited thereto.

In the present application, the disease or pathological condition isselected from inflammatory diseases, autoimmune diseases, diseasesaccompanied by tissue damage, ischemia, or necrosis, and fibroticdiseases, but is not limited thereto.

In the present application, the inflammatory disease or autoimmunedisease is, for example, selected from inflammatory bowel disease andpsoriasis, but is not limited thereto. The fibrotic disease is selectedfrom, for example, lung fibrosis, liver fibrosis, and liver cirrhosis,but is not limited thereto. The disease accompanied by tissue damage,ischemia, or necrosis includes, for example, inflammatory bowel disease,but is not limited thereto. The inflammatory bowel disease includes, butis not limited to, ulcerative colitis and Crohn's disease.

In the present application, the term “nuclear protein” refers to aprotein that exerts a certain function in the nucleus and is a proteinother than 1) to 6) below:

1) High mobility group box 1 (HMGB1) protein;2) High mobility group box 2 (HMGB2) protein;3) High mobility group box 3 (HMGB3) protein;4) S100 calcium-binding protein A8 (S100A8) protein;5) S100 calcium-binding protein A9 (S100A9) protein; and6) Interleukin-1 (IL-1) family cytokines. In one embodiment, the nuclearprotein of the present application is a protein that has the activity ofmobilizing mesenchymal stem cells into peripheral blood.

In the present application, the term “activity of mobilizing mesenchymalstem cells into peripheral blood” is used interchangeably with “activityto increase the abundance of mesenchymal stem cells in peripheralblood”.

The nuclear protein in the present application includes, but is notlimited to, for example, a nuclear protein involved in transcriptionalregulation. A “protein involved in transcriptional regulation” refersto, among nuclear proteins, a protein having a function of regulatingany process in transcription and includes, for example, transcriptionfactors and transcription cofactors, but is not limited thereto.

In the present application, the transcription factor is a protein thatcontrols transcription by binding to DNA by itself or in the form of acomplex with other proteins, and includes general transcription factors(proteins that constitute a transcription apparatus), transcriptionalregulation factors, transcription elongation factors, factors thatregulate transcription by involvement in the transcription terminationprocess, and such.

In the present application, the transcription cofactor is a protein thatregulates transcription via protein-protein interactions without bindingdirectly to DNA, and includes but is not limited to co-activators andco-repressors, which regulate transcription by intervening between(binding to both) a transcriptional regulatory factor and a generaltranscription factor.

In the present application, the “fragment peptide of a nuclear protein”refers to a fragment peptide derived from the above-mentioned nuclearprotein. In one embodiment, a fragment peptide of a nuclear protein is afragment that has an activity of mobilizing mesenchymal stem cells intothe peripheral blood.

Fragment peptides of the HMGB1 protein, fragment peptides of the HMGB2protein, fragment peptides of the HMGB3 protein, fragment peptides ofthe S100A8 protein, fragment peptides of the S100A9 protein, andfragment peptides of the IL-1 family cytokines are excluded from thefragment peptides of nuclear proteins in the present application.

In the present application, the term “a fragment peptide of a nuclearprotein” is used interchangeably with “a fragment peptide derived from anuclear protein”, “a partial peptide derived from a nuclear protein”, “afragment peptide consisting of a portion of a nuclear protein”, “apartial peptide consisting of a portion of a nuclear protein”, or “apartial peptide of a nuclear protein”.

The activity of a nuclear protein or a fragment peptide thereof in thepresent application to mobilize mesenchymal stem cells into theperipheral blood can be assessed by i) collecting peripheral blood froman individual administered with a nuclear protein or a fragment peptidethereof and an individual not administered with the nuclear protein orfragment peptide, seeding and culturing in a culture dish (several daysto 10 days), and counting the number of colonies formed; and ii)confirming that the formed colonies have the ability to adhere to thesolid phase and proliferate (self-renewal ability), and the ability todifferentiate into osteoblasts, chondrocytes and adipocytes. In i)above, before seeding the collected peripheral blood on a culture dish,red blood cells may be removed from the peripheral blood in a desiredmanner.

The nuclear protein or fragment peptide thereof in the presentapplication can be obtained as a recombinant by incorporating DNAencoding it into an appropriate expression system, or it can beartificially synthesized. Thus, nuclear proteins or fragment peptidesthereof in this application include nuclear proteins and fragmentpeptides thereof prepared using cells, and artificially synthesizednuclear proteins and fragment peptides thereof (i.e., artificial(synthetic) nuclear proteins and fragment peptides thereof).

In order to obtain the nuclear protein or fragment peptide thereof inthe present application by genetic engineering techniques, DNA encodingthe peptide may be incorporated into an appropriate expression systemand expressed.

Hosts applicable to the present application include, but are not limitedto, prokaryotic cells and eukaryotic cells. In addition, hostsapplicable to the present application also include bacteria (e.g.,Escherichia coli), yeast, animal cells (e.g., mammalian cells such asHEK293 cells and CHO cells, insect cells such as silkworm cells), plantcells and such, but are not limited thereto.

As the host/vector system applicable to the present application, forexample, the expression vector pGEX and Escherichia coli can be shown.pGEX can express a foreign gene as a fusion protein with glutathioneS-transferase (GST) (Gene, 67: 31-40, 1988). As such, pGEX into whichDNA encoding the nuclear protein or fragment peptide thereof of thepresent application has been incorporated is introduced into an E. colistrain such as BL21 by heat shock, and after culturing for anappropriate period of time, isopropylthio-β-D-galactoside (IPTG) isadded to induce the expression of the GST fusion peptide. GST in thepresent application adsorbs to glutathione Sepharose 4B, and thus theexpression product can be easily separated and purified by affinitychromatography.

In addition to this, the following can be applied as a host/vectorsystem for obtaining a genetic recombinant of the nuclear protein orfragment peptide thereof of the present application. First, when abacterium is used as a host, a vector for expressing a fusion proteinusing a tag or such is commercially available. In addition, the geneticrecombinant of the present application also includes those in which atag or a partial peptide thereof is added.

The tag added to the nuclear protein or fragment peptide thereof of thepresent application is not particularly limited as long as it does notaffect the activity of the nuclear protein or fragment peptide thereofof the present application, and includes, for example, a histidine tag(for example, 6×His, 10×His), HA tag, FLAG tag, GST tag, T7-tag,HSV-tag, E-tag, lck tag, and B-tag.

Among yeasts, it is known that Pichia yeast is effective for theexpression of proteins having sugar chains. In terms of the addition ofsugar chains, the expression system that uses a Baculovirus vector,which uses an insect cell as a host, is also useful (Bio/Technology,6:47-55, 1988). Furthermore, mammalian cells are used for transfectionwith a vector that uses the promoter of CMV, RSV, SV40, or such. Thesehost/vector systems can be used as an expression system for the nuclearproteins or fragment peptides thereof of the present application. Inaddition, plasmid vectors, retrovirus vectors, lentivirus vectors,adenovirus vectors, adeno-associated virus vectors, Sendai virusvectors, Sendai virus envelope vectors, papilloma virus vectors, andsuch virus vectors may also be used to introduce the gene, withoutlimitation thereto. The vector may also contain a promoter DNA sequencethat effectively induces gene expression, factors that control geneexpression, and molecules necessary to maintain the stability of theDNA.

The resulting nuclear proteins or fragment peptides thereof in thepresent application can be isolated from the host cell or outside of thecell (such as medium), and purified as a substantially pure homogeneousprotein. For separation and purification of proteins, any separation andpurification methods used in standard protein purification may beutilized, without limitation. For example, chromatography columns,filters, ultrafiltration, salting out, solvent precipitation, solventextraction, distillation, immunoprecipitation, SDS-polyacrylamide gelelectrophoresis, isoelectric focusing, dialysis, recrystallization, andsuch are appropriately selected and combined for protein separation andpurification.

Chromatography includes, for example, affinity chromatography, ionexchange chromatography, hydrophobic chromatography, gel filtration,reverse phase chromatography, adsorption chromatography and such(Marshak et al, Strategies for Protein Purification andCharacterization: A Laboratory Course Manual. Ed Daniel R. Cold SpringHarbor Laboratory Press, 1996). These chromatographies can be performedusing liquid chromatography such as HPLC and FPLC.

Further, the nuclear protein or fragment peptide thereof in the presentapplication is preferably a substantially purified peptide. Here,“substantially purified” means that the degree of purification of thenuclear protein or fragment peptide thereof of the present application(the ratio of the nuclear protein or fragment peptide thereof of thepresent application in the entire protein component) is 50% or more, 60%or more, 70% or more, 80% or more, 90% or more, 95% or more, 100% ornearly 100%. The nearly 100% upper limit depends on the purificationtechniques and analysis techniques of those skilled in the art and maybe, for example, 99.999%, 99.99%, 99.9%, or 99%.

Further, the substantially purified nuclear protein or fragment peptidethereof includes those purified by whatever purification method as longas they have the above purity. Examples include, but are not limited to,nuclear proteins and fragment peptides thereof substantially purified byappropriately selecting or combining the above-mentioned chromatographycolumns, filters, ultrafiltration, salting out, solvent precipitation,solvent extraction, distillation, immunoprecipitation,SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis,recrystallization and such.

On the other hand, the nuclear proteins or fragment peptides thereof inthe present application can also be artificially synthesized. In thepeptide synthesis method of the present application, peptides can bechemically synthesized by methods such as a peptide liquid-phasesynthesis method and a peptide solid-phase synthesis method. The peptidesolid-phase synthesis method is one of the methods generally used whenchemically synthesizing a peptide. Polystyrene polymer gel beads havinga diameter of about 0.1 mm modified with amino groups on the surface areused as a solid phase, from which the amino acid chain is extended oneby one via dehydration reaction. When the sequence of the target peptideis completed, it is excised from the solid phase surface to obtain thetarget substance. Solid phase synthesis enables synthesis of ribosomepeptides, which are difficult to synthesize in bacteria, introduction ofunnatural amino acids such as D-form and stable isotope (²H, ¹³C, ¹⁵N,etc.)-substituted amino acids, introduction of heavy atom-substitutedamino acids (e.g., selenoamino acids such as selenomethionine),modification of peptide and protein main chains, and such. Whensynthesizing a long peptide chain of 70 to more than 100 amino acids inthe solid phase method, it can be synthesized by ligating two peptidechains using the native chemical ligation method. The nuclear protein orfragment peptide thereof in the present application may be in the formof a pharmaceutically acceptable salt of the protein or peptide.Examples of pharmaceutically acceptable salts include, but are notlimited to, hydrochlorides, acetates, and trifluoroacetates. The nuclearprotein or fragment peptide thereof in the present application may be inthe form of a solvate of the protein or peptide, or a solvate of apharmaceutically acceptable salt of the protein or peptide. A solvaterefers to a solute molecule to which an arbitrary number of solventmolecules are coordinated, and examples thereof include hydrates, butare not limited thereto.

The amino acid length of the nuclear protein or fragment peptide thereofin the present application includes, for example, 25 to 35 amino acids,20 to 40 amino acids, 10 to 50 amino acids, 10 to 70 amino acids, and 10to 100 amino acids, but is not limited thereto.

Examples of the nuclear protein or fragment peptide thereof in thepresent application include nuclear proteins selected from below orfragment peptides derived therefrom:

1. BTF3 protein (Basic transcription factor 3);2. SUPT16H protein (Suppressor of Ty 16 Homolog; or Facilitateschromatin transcription complex subunit SPT16);3. YBX1 protein (Y-Box binding protein 1; or Nuclease-sensitiveelement-binding protein 1);4. NPM1 protein (Nucleophosmin 1);5. PA2G4 protein (Proliferation-associated protein 2G4);6. PFDN5 protein (Prefoldin subunit 5);7. PSMC3 protein (Proteasome (Prosome, Macropain) 26S subunit, ATPase 3;or 26S proteasome regulatory subunit 6A);8. HNRNPK protein (Heterogeneous nuclear ribonucleoprotein K); and9. Nuclear protein functionally equivalent to a protein selected from 1to 8.

In light of the examples of the fragment peptides described in theExamples of the present application, the nuclear protein selected from 1to 8 above is considered to have an activity of mobilizing mesenchymalstem cells to peripheral blood. Therefore, “functionally equivalent” asdescribed in 9 above means functionally equivalent in terms of theactivity of mobilizing mesenchymal stem cells to peripheral blood.Therefore, the nuclear protein described in 9 above can be expressed asa nuclear protein having an activity equivalent to that of the proteinselected from 1 to 8 (equivalent activity to mobilize mesenchymal stemcells to peripheral blood). The fragment peptide derived from thenuclear protein selected from 1 to 9 above is a fragment peptide havingan activity of mobilizing mesenchymal stem cells to peripheral blood.

Since the nuclear protein selected from 1 to 9 above or a fragmentpeptide thereof has an activity of mobilizing mesenchymal stem cells toperipheral blood, it is considered to have the effect of mobilizingmesenchymal stem cells to peripheral blood, as well as therapeuticeffects on inflammatory diseases, autoimmune diseases, diseasesaccompanied by tissue damage, ischemia, or necrosis, and fibroticdiseases.

In the present application, the nuclear protein or a fragment peptidethereof includes, for example, a nuclear protein selected from below ora fragment peptide derived therefrom:

(I) a nuclear protein comprising an amino acid sequence selected fromSEQ ID NOs: 1 to 34;(II) a nuclear protein consisting of an amino acid sequence selectedfrom SEQ ID NOs: 1 to 34;(III) a nuclear protein comprising an amino acid sequence resulting fromsubstitution, deletion, insertion, or addition of one or more aminoacids in an amino acid sequence selected from SEQ ID NOs: 1 to 34;(IV) a nuclear protein consisting of an amino acid sequence resultingfrom substitution, deletion, insertion, or addition of one or more aminoacids in an amino acid sequence selected from SEQ ID NOs: 1 to 34;(V) a nuclear protein comprising an amino acid sequence having about 80%or more sequence identity with an amino acid sequence selected from SEQID NOs: 1 to 34;(VI) a nuclear protein consisting of an amino acid sequence having about80% or more sequence identity with an amino acid sequence selected fromSEQ ID NOs: 1 to 34;(VII) a nuclear protein encoded by a DNA consisting of a nucleotidesequence selected from SEQ ID NOs: 57 to 90; and(VIII) a nuclear protein encoded by a DNA that hybridizes understringent conditions with a DNA consisting of a nucleotide sequenceselected from SEQ ID NOs: 57 to 90.

In the present application, the fragment peptide of a nuclear proteinincludes, for example, a fragment peptide selected from below:

(i) a nuclear protein fragment peptide consisting of a portion of anamino acid sequence selected from SEQ ID NOs: 1 to 34;(ii) a nuclear protein fragment peptide comprising an amino acidsequence selected from SEQ ID NOs: 35-56;(iii) a nuclear protein fragment peptide consisting of an amino acidsequence selected from SEQ ID NOs: 35 to 56;(iv) a nuclear protein fragment peptide consisting of a portion of anamino acid sequence selected from SEQ ID NOs: 35 to 56;(v) a nuclear protein fragment peptide comprising an amino acid sequenceresulting from substitution, deletion, insertion, or addition of one ormore amino acids in an amino acid sequence selected from SEQ ID NOs: 35to 56;(vi) a nuclear protein fragment peptide consisting of an amino acidsequence resulting from substitution, deletion, insertion, or additionof one or more amino acids in an amino acid sequence selected from SEQID NOs: 35 to 56;(vii) a nuclear protein fragment peptide comprising an amino acidsequence having about 80% or more sequence identity with an amino acidsequence selected from SEQ ID NOs: 35 to 56;(viii) a nuclear protein fragment peptide consisting of an amino acidsequence having about 80% or more sequence identity with an amino acidsequence selected from SEQ ID NOs: 35 to 56;(ix) a nuclear protein fragment peptide encoded by a DNA consisting of anucleotide sequence selected from SEQ ID NOs: 91 to 112; and(x) a nuclear protein fragment peptide encoded by a DNA that hybridizesunder stringent conditions with a DNA consisting of a nucleotidesequence selected from SEQ ID NOs: 91 to 112.

In the present application, “plurality” includes, for example, 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.

In the present application, “about 80% or more” means, for example,about 85% or more, about 90% or more, about 91% or more, about 92% ormore, about 93% or more, about 94% or more, about 95% or more, about 96%or more, about 97% or more, about 98% or more, or about 99% or more.

In the present application, the “stringent conditions” can be shown asconditions of, for example, hybridization at 6×SSC, 40% formamide, 25°C., and washing at 1×SSC, 55° C. Stringency is affected by conditionssuch as salt concentration, formamide concentration, or temperature, andthose skilled in the art can set these conditions to obtain the requiredstringency.

When the hybridization is carried out under stringent conditions, a DNAhaving a high homology in terms of nucleotide sequence is selected, andthe possibility is increased for the protein isolated as a result tocomprise a protein functionally equivalent to (e.g., homologue) aprotein consisting of an amino acid sequence selected from SEQ ID NOs: 1to 34, or to comprise a fragment peptide functionally equivalent to afragment peptide consisting of an amino acid sequence selected from SEQID NOs: 35 to 56. A nucleotide sequence having high homology canexhibit, for example, about 60% or more, about 70% or more, or about 80%or more identity.

Further, in the present application, the fragment peptide of a nuclearprotein includes, for example, a fragment peptide selected from below:

(1) a fragment peptide consisting of a portion of an amino acid sequenceselected from SEQ ID NOs: 1 to 2, and which is a fragment peptidecomprising the amino acid sequence described in SEQ ID NO: 35;(2) a fragment peptide consisting of a portion of the amino acidsequence described in SEQ ID NO: 3, and which is a fragment peptidecomprising the amino acid sequence described in SEQ ID NO: 36;(3) a fragment peptide consisting of a portion of the amino acidsequence described in SEQ ID NO: 4, and which is a fragment peptidecomprising an amino acid sequence selected from SEQ ID NOs: 37 to 39;(4) a fragment peptide consisting of a portion of an amino acid sequenceselected from SEQ ID NOs: 5 to 7, and which is a fragment peptidecomprising an amino acid sequence selected from SEQ ID NOs: 40 to 41;(5) a fragment peptide consisting of a portion of the amino acidsequence described in SEQ ID NO: 8, and which is a fragment peptidecomprising the amino acid sequence described in SEQ ID NO: 42;(6) a fragment peptide consisting of a portion of the amino acidsequence described in SEQ ID NO: 9, and which is a fragment peptidecomprising the amino acid sequence described in SEQ ID NO: 43;(7) a fragment peptide consisting of a portion of the amino acidsequence described in SEQ ID NO: 10, and which is a fragment peptidecomprising the amino acid sequence described in SEQ ID NO: 44;(8) a fragment peptide consisting of a portion of an amino acid sequenceselected from SEQ ID NOs: 11 to 16, and which is a fragment peptidecomprising the amino acid sequence described in SEQ ID NO: 45;(9) a fragment peptide consisting of a portion of an amino acid sequenceselected from SEQ ID NOs: 17 to 18, and which is a fragment peptidecomprising the amino acid sequence described in SEQ ID NO: 46;(10) a fragment peptide consisting of a portion of the amino acidsequence described in SEQ ID NO: 19, and which is a fragment peptidecomprising the amino acid sequence described in SEQ ID NO: 47;(11) a fragment peptide consisting of a portion of the amino acidsequence described in SEQ ID NO: 20, and which is a fragment peptidecomprising an amino acid sequence selected from SEQ ID NOs: 48 to 50;(12) a fragment peptide consisting of a portion of an amino acidsequence selected from SEQ ID NOs: 21, 23, and 24, and which is afragment peptide comprising the amino acid sequence described in SEQ IDNO: 51;(13) a fragment peptide consisting of a portion of an amino acidsequence selected from SEQ ID NOs: 21 to 26, and which is a fragmentpeptide comprising the amino acid sequence described in SEQ ID NO: 52;(14) a fragment peptide consisting of a portion of the amino acidsequence described in SEQ ID NO: 27, and which is a fragment peptidecomprising the amino acid sequence described in SEQ ID NO: 53;(15) a fragment peptide consisting of a portion of an amino acidsequence selected from SEQ ID NOs: 28 to 29, and which is a fragmentpeptide comprising the amino acid sequence described in SEQ ID NO: 54;(16) a fragment peptide consisting of a portion of the amino acidsequence described in SEQ ID NO: 30, and which is a fragment peptidecomprising the amino acid sequence described in SEQ ID NO: 55; and(17) a fragment peptide consisting of a portion of an amino acidsequence selected from SEQ ID NOs: 31 to 34, and which is a fragmentpeptide comprising the amino acid sequence described in SEQ ID NO: 56.

The nuclear protein selected from (I) to (VIII) above is a nuclearprotein having an activity of mobilizing mesenchymal stem cells intoperipheral blood. Further, the fragment peptides derived from a nuclearprotein selected from (I) to (VIII) above, nuclear protein fragmentpeptides selected from (i) to (x) above, and nuclear protein fragmentpeptides selected from (1) to (17) above are fragment peptides having anactivity of mobilizing mesenchymal stem cells into peripheral blood.Therefore, these nuclear proteins and fragment peptides are consideredto have the effect of mobilizing mesenchymal stem cells into peripheralblood, as well as therapeutic effects on inflammatory diseases,autoimmune diseases, diseases accompanied by tissue damage, ischemia, ornecrosis, and fibrotic diseases.

The present application also provides nuclear proteins selected from 1to 9 above or fragment peptides derived therefrom, nuclear proteinsselected from (I) to (VIII) above or fragment peptides derivedtherefrom, nuclear protein fragment peptides selected from (i) to (x)above, and nuclear protein fragment peptides selected from (1) from (17)above.

The amino acid sequences described in SEQ ID NOs: 1 to 56 are amino acidsequences of the proteins or peptides shown in Tables 1-1 and 1-2 below.

TABLE 1-1 SEQ Position in the ID NO: Name full-length protein 1 MouseBTF3 protein isoform 1 2 Mouse BTF3 protein isoform 2 3 Mouse SUPT16Hprotein 4 Mouse YBX1 protein 5 Mouse NPM1 protein isoform 1 6 Mouse NPM1protein isoform 2 7 Mouse NPM1 protein isoform 3 8 Mouse PA2G4 protein 9Mouse PFDN5 protein 10 Mouse PSMC3 protein 11 Mouse HNRNPK proteinisoform 1 12 Mouse HNRNPK protein isoform 2 13 Mouse HNRNPK proteinisoform 3 14 Mouse HNRNPK protein isoform 4 15 Mouse HNRNPK proteinisoform 5 16 Mouse HNRNPK protein isoform 6 17 Human BTF3 proteinisoform A 18 Human BTF3 protein isoform B 19 Human SUPT16H protein 20Human YBX1 protein 21 Human NPM1 protein isoform 1 22 Human NPM1 proteinisoform 2 23 Human NPM1 protein isoform 3 24 Human NPM1 protein isoform4 25 Human NPM1 protein isoform 5 26 Human NPM1 protein isoform 6 27Human PA2G4 protein

TABLE 1-2 28 Human PFDN5 protein isoform α 29 Human PFDN5 proteinisoform γ 30 Human PSMC3 protein 31 Human HNRNPK protein isoform a 32Human HNRNPK protein isoform b 33 Human HNRNPK protein isoform c 34Human HNRNPK protein isoform d 35 Mouse BTF3 peptide-1 63-92 36 MouseSUPT16H peptide-1 108-137 37 Mouse YBX1 peptide-1 174-203 38 Mouse YBX1peptide-2 225-254 39 Mouse YBX1 peptide-3 273-302 40 Mouse NPM1peptide-1 209-238 41 Mouse NPM1 peptide-2 221-250 42 Mouse PA2G4peptide-1 43-72 43 Mouse PFDN5 peptide-1  99-128 44 Mouse PSMC3peptide-1 55-84 45 Mouse HNRNPK peptide-1 242-271 46 Human BTF3peptide-1 65-94 47 Human SUPT16H peptide-1 108-137 48 Human YBX1peptide-1 176-205 49 Human YBX1 peptide-2 227-256 50 Human YBX1peptide-3 275-304 51 Human NPM1 peptide-1 210-240 52 Human NPM1peptide-2 223-252 53 Human PA2G4 peptide-1 43-72 54 Human PFDN5peptide-1  99-128 55 Human PSMC3 peptide-1 52-81 56 Human HNRNPKpeptide-1 242-271

The nucleotide sequences described in SEQ ID NOs: 57 to 112 are examplesof the nucleotide sequences of DNAs encoding the proteins or peptidesshown in Tables 2-1 and 2-2 below. Other DNA sequences encoding theproteins or peptides shown in Tables 2-1 and 2-2 below can be producedby a method of converting the amino acid residues of the proteins orpeptides to corresponding codons using codon tables known to thoseskilled in the art (reverse translation). Reverse translation can beperformed by using a variety of software (including programs,algorithms, etc.) developed for the analysis of amino acid and nucleicacid sequences as desired.

TABLE 2-1 SEQ Position in ID NO: Name full length DNA 57 Mouse BTF3protein isoform 1 58 Mouse BTF3 protein isoform 2 59 Mouse SUPT16Hprotein 60 Mouse YBX1 protein 61 Mouse NPM1 protein isoform 1 62 MouseNPM1 protein isoform 2 63 Mouse NPM1 protein isoform 3 64 Mouse PA2G4protein 65 Mouse PFDN5 protein 66 Mouse PSMC3 protein 67 Mouse HNRNPKprotein isoform 1 68 Mouse HNRNPK protein isoform 2 69 Mouse HNRNPKprotein isoform 3 70 Mouse HNRNPK protein isoform 4 71 Mouse HNRNPKprotein isoform 5 72 Mouse HNRNPK protein isoform 6 73 Human BTF3protein isoform A 74 Human BTF3 protein isoform B 75 Human SUPT16Hprotein 76 Human YBX1 protein 77 Human NPM1 protein isoform 1 78 HumanNPM1 protein isoform 2 79 Human NPM1 protein isoform 3 80 Human NPM1protein isoform 4 81 Human NPM1 protein isoform 5 82 Human NPM1 proteinisoform 6 83 Human PA2G4 protein 84 | Human PFDN5 protein isoform α

TABLE 2-2 85 Human PFDN5 protein isoform γ 86 Human PSMC3 protein 87Human HNRNPK protein isoform a 88 Human HNRNPK protein isoform b 89Human HNRNPK protein isoform c 90 Human HNRNPK protein isoform d 91Mouse BTF3 peptide-1 187-276 92 Mouse SUPT16H peptide-1 322-411 93 MouseYBX1 peptide-1 520-609 94 Mouse YBX1 peptide-2 673-762 95 Mouse YBX1peptide-3 817 906 96 Mouse NPM1 peptide-1 625-714 97 Mouse NPM1peptide-2 661-750 98 Mouse PA2G4 peptide-1 127-216 99 Mouse PFDN5peptide-1 295-384 100 Mouse PSMC3 peptide-1 163-252 101 Mouse HNRNPKpeptide-1 724-813 102 Human BTF3 peptide-1 193-282 103 Human SUPT16Hpeptide-1 322-411 104 Human YBX1 peptide-1 526-615 105 Human YBX1peptide-2 679-768 106 Human YBX1 peptide-3 823-912 107 Human NPM1peptide-1 628-720 108 Human NPM1 peptide-2 667-756 109 Human PA2G4peptide-1 127-216 110 Human PFDN5 peptide-1 295-384 111 Human PSMC3peptide-1 154-243 112 Human HNRNPK peptide-1 724-813

In the present application, the nuclear protein or a fragment peptidethereof is a nuclear protein or a fragment peptide thereof thatcomprises, for example, a nuclear localization signal (NLS). A nuclearlocalization signal (NLS) is an amino acid sequence having a certainpattern and has a function of transferring a protein/peptide having theamino acid sequence into the nucleus.

For example, many nuclear proteins are known to carry NLS in their aminoacid sequences, and move into the nucleus by binding to the nucleartransport receptor (also referred to as nuclear transport proteins andnuclear transport factors) that recognizes the NLS.

Examples of currently known NLS (hereinbelow referred to as a known NLS)include those listed in Tables 3-1 and 3-2 below.

TABLE 3-1 Type Sequence pattern Specific example (source) cNLSmonopartite K-K/R-X-K/R (SEQ ID PKKKRRV (classical NO: 113)(Lange et al., (2007) J. Biol. NLS) Chem., 282, 5101-5105;Kosugi et al., (2009) J. Biol. Chem., 284, 478-485., etc.)(SEQ ID NO: 114) bipartite (K/R)(K/R)X₁₀₋₁₂(K/R)_(3/5) KRPAATKKAGQAKKKK((K/R)_(3/5 )= at least 3 amino (Kosugi et at., (2009) J. Biol.acids among 5 consecutive Chem., 284, 478-485.,amino acids are K or R) (SEQ etc.) (SEQ ID NO: 118) ID NO: 115)KRX₁₀₋₁₂K(K/R)(K/R) (SEQ ID NO: 116) KRX₁₀₋₁₂K(K/R)X(K/R) (SEQID NO: 117) PY-NLS Those having the following 2 FGNYNNQSSNFGPMKGGNFmotifs: GGRSSGPY 1) R/K/H-X₍₂₋₅₎-P-Y (SEQ ID(Lee et al., (2006) Cell, 126, NO: 119) 543-558; Süel et al., (2008) P2) φG/A/Sφφ X₍₁₁₋₁₃₎PY LoS Biol., 6, e137, etc.) (SEQφ is an amino acid having ID NO: 121) hydrophobicside-chain) (SEQ ID NO: 120) Those having the following 2GEGERPAQNEKRKEKNIKRG motifs: GNRFEPY 1) R/K/H-X₍₂₋₅₎-P-Y (SEQ ID(Lee et al., (2006) Cell, 126, NO: 119) 543-558; Süel et al., (2008)2) basic-enriched₍₅₀₈₎X₍₈₋₁₀₎PY PLoS Biol., 6, e137, etc.)(“basic-enriched” is a region (SEQ ID NO: 123) rich in basic aminoacids) (SEQ ID NO: 122)

TABLE 3-2 BIB domain Amino acid residues 32-74 VHSHKKKKIRTSPTFRRPKTL(β-like importin binding of rpL23a protein RLRRQPKYPRKSAPRRNKLD domain)HY (Jäkel, S. & Görlich, D. (1998) EMBO J., 17, 4491-4502) (SEQID NO: 124) BIB domain like sequence (1) MSHRKFSAPRHGSLGKimura et al., Mol. Cell.     FLPRKRSSRHRGKVKS Proteomics (2013), 12,    FPKDDP (SEQ ID NO: 145-157, FIG. 3C 2)-3)     125)(2) EVTNDFVMLKGCVVG     TKKRVLTLRKSLLVQTK     RRALEKIDLKFIDTTSK    F (SEQ ID NO: 126) (3) SLGQSASETEEDTVSV     SKKEKNRKRRNRKKK    KKPQRVRGVSSESSG     DREK (SEQ ID NO: 127) (4) PTRYSVDIPLDKTVVN    KDVFRDPALKRKARRE     AKVKFEERYKTGKNK     WFF (SEQ ID NO: 128)(5) KMFKGKRGAQLAKDIA     RRSKTFNPGAGLPTDK     KKGGPSPGDVEAIKNA    IA (SEQ ID NO: 129)

In addition, known NLS include sequences registered on the NLSdbdatabase (https://rostlab.org/services/nlsdb/). The sequences registeredon NLSdb can be viewed and downloaded on the above website. Among theNLS sequences registered on NLSdb, those whose annotation type is“Experimental” or “By Expert” can be estimated as having a function oftranslocating a protein/peptide into the nucleus, and are thereforetreated as known NLS in the present application.

The NLS in the present application may be an NLS predicted by using aspecific program (hereinbelow referred to as a predicted NLS). Whether apredicted NLS is contained in the desired amino acid sequence can bedetermined using the following program: SeqNLS (Lin et al., PLoS One.2013 Oct. 29; 8 (10):e76864) or NLStradamus (Nguyen et al., BMCBioinformatics. 2009 Jun. 29; 10:202).

In one embodiment, the NLS is a known NLS. In one embodiment, the NLS isa known NLS selected from the group consisting of cNLS, PY-NLS, BIBdomain, and BIB domain-like sequences. In one embodiment, the cNLS is amonopartite cNLS. In one embodiment, the monopartite cNLS is KKEK (SEQID NO: 130).

An effective amount of a nuclear protein of the present application or afragment peptide thereof, or a pharmaceutical composition comprising thesame (hereinafter referred to as a pharmaceutical composition and such)is administered to a subject for the treatment of diseases andconditions described herein.

The effective amount in the present application means an amountsufficient for the treatment of the diseases or pathological conditionsdescribed herein. The treatment in the present application includesalleviation, delay, inhibition, amelioration, remission, cure, and fullrecovery, but are not limited thereto.

There is no limitation on the site of administration of thepharmaceutical composition and such of the present application, and thepharmaceutical composition and such of the present application can exertits effect when administered to any site, such as a site where thesymptoms of the disease or pathological condition appear or a sitenearby, a site different from these sites (sites other than thesesites), a site separated from a site where the symptoms of the diseaseor pathological condition appear, a site distal to a site where thesymptoms of the disease or pathological condition appear, or a sitedistal and ectopic to a site where the symptoms of the disease orpathological condition appear.

Further, the pharmaceutical composition and such of the presentapplication can exert its effect when administered to any tissue, suchas a tissue different from a tissue in which the symptoms of the diseaseor the pathological condition appear, a tissue separated from a tissuein which the symptoms of the disease or the pathological conditionappear, a tissue distal to a tissue in which the symptoms of the diseaseor the pathological condition appear, or a tissue distal and ectopic toa tissue in which the symptoms of the disease or pathological conditionappear.

Methods of administering the pharmaceutical composition and such of thepresent application include, but are not limited to, oral administrationand parenteral administration, and methods of parenteral administrationinclude intravascular administration (intra-arterial administration,intravenous administration, etc.), intramuscular administration,subcutaneous administration, intradermal administration, intraperitonealadministration, nasal administration, pulmonary administration,transdermal administration, and such. In addition, the pharmaceuticalcomposition and such of the present application can be administeredsystemically or locally (for example, subcutaneously, intradermally, orto the skin surface, eyeball, or palpebral conjunctiva, nasal mucosa,oral and gastrointestinal mucosa, vaginal and endometrial mucosa, orinjured site) by injection administration, for example, intravenousinjection, intramuscular injection, intraperitoneal injection, andsubcutaneous injection.

In place of the nuclear protein of the present application or a fragmentpeptide thereof, a cell secreting the nuclear protein or a fragmentpeptide thereof, a gene therapy vector into which a DNA encoding thenuclear protein or fragment peptide thereof is inserted, and apharmaceutical composition comprising them can be used.

In addition, the administration method can be appropriately selecteddepending on the age and symptoms of a patient. When the pharmaceuticalcomposition and such of the present application is administered, forexample, the dose can be selected from the range of 0.0000001 mg to 1000mg per kilogram of body weight per administration. Alternatively, forexample, the dose can be selected from the range of 0.00001 to 100000mg/body per patient. When administering cells secreting the nuclearprotein of the present application or a fragment peptide thereof or genetherapy vectors into which DNA encoding the nuclear protein or afragment peptide thereof is inserted, they can be administered so thatthe amount of the nuclear protein or fragment peptide thereof is withinthe above range. However, the pharmaceutical compositions in the presentapplication are not limited to these dosages.

The pharmaceutical compositions of the present application can beformulated according to conventional methods (e.g., Remington'sPharmaceutical Science, latest edition, Mark Publishing Company, Easton,U.S.A.), and may contain pharmaceutically acceptable carriers oradditives together. Examples include, but are not limited to,surfactants, excipients, coloring agents, perfumes, preservatives,stabilizers, buffers, suspending agents, isotonizing agents, bindingagents, disintegrants, lubricants, fluidity-promoting agents, andflavoring agents. Other commonly used carriers can also be used asappropriate. Specific examples include light anhydrous silicic acid,lactose, crystalline cellulose, mannitol, starch, carmellose calcium,carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone,gelatin, medium-chain fatty acid triglyceride, polyoxyethylenehydrogenated castor oil 60, white sugar, carboxymethyl cellulose,cornstarch, and inorganic salts.

All prior art documents cited herein are incorporated herein asreferences.

Herein below, the present invention will be further illustrated withreference to Examples, but it is not to be construed as being limitedthereto.

EXAMPLES Example 1 Mobilization of Mesenchymal Stem Cells by FragmentPeptides of Nuclear Proteins (1) Materials and Methods i) PeptideProduction

Fragment peptides of the nuclear proteins shown in the table below werechemically synthesized by the solid phase method (all of the obtainedpeptides are in the form of trifluoroacetic acid (TFA) salts).

TABLE 4 Another name considering the Name name of the protein origin SEQID NO NP-1 Mouse BTF3 peptide-1 35 NP-2 Mouse SUPT16H peptide-1 36 NP-3Mouse YBX1 peptide-1 37 NP-4 Mouse YBX1 peptide-2 38 NP-5 Mouse YBX1peptide-3 39 NP-6 Mouse NPM1 peptide-1 40 NP-7 Mouse NPM1 peptide-2 41NP-8 Mouse PA2G4 peptide-1 42 NP-9 Mouse PFDN5 peptide-1 43 NP-10 MousePSMC3 peptide-1 44 NP-11 Mouse HNRNPK peptide-1 45

ii) Peptide Administration

C57BL/6J mice (8 weeks old, male, body weight 25 g) were prepared anddivided into groups to which any of peptides NP-1 to NP-11 described inthe above table were administered and a control group. The peptide wasadministered by injecting into the tail vein, a solution of each peptideadjusted to a concentration of 1 μg/μL using physiological saline as asolvent in an amount of 100 μL/animal (4 mg/kg dose of peptide). For thecontrol group, physiological saline was injected into the tail vein inan amount of 100 μL/animal.

iii) Cell Collection from Peripheral Blood

Predetermined time after the administration of physiological saline orpeptides NP-1 to NP-11 (NP-1 to NP-6 and NP-8: after 14 hours; NP-7,NP-10 and NP-11: after 16 hours; NP-9: after 24 hours), about 800 μL to1000 μL of peripheral blood was collected from the hearts under generalanesthesia (1 mL syringe containing heparin was used). To remove redblood cells, an equal volume of Hetasep (STEMCELL Technologies Inc., CatNo. ST-07906) as the collected blood was added and centrifuged for 2 minat 100G, incubated for 15 min at room temperature, and then thesupernatant was collected. The supernatant was used in the nextexperiment as a sample containing nucleated cells in peripheral blood.

iv) Colony Assay

The supernatant (sample containing peripheral blood-derived cells)obtained by the above procedure was seeded on collagen I-coated 6-wellplates (Corning, Cat No. 356400), and cultured for 10 days under theconditions of 37° C., 5% CO₂ and 5% O₂, using a medium containingExpansion Medium prepared using the MesenCult Expansion Kit (STEMCELLTechnologies, Cat No. ST-05513) according to the manual of the kit, 1%L-glutamine (Nacalai Tesque Inc.), 10 μM ROCK inhibitor (Y27632, TocrisBioscience) and 1% penicillin/streptomycin (Nacalai Tesque Inc.) (allnumerical values are final concentrations). The medium was replaced withfresh medium twice a week during the culture period. On the 10^(th) dayof culture, cells on the plates were stained with a Differential QuikStain Kit (Sysmex Corporation, Cat No. 16920), and the number ofcolonies containing 50 or more cells was counted.

In the experiments conducted by the present inventors so far, all thecolonies obtained as a result of culturing peripheral blood on a solidphase such as a dish or a plate have adherability to the solid phase andhave self-renewal ability. In addition, they have been confirmed to bePDGFRα positive, and have the ability to differentiate into bone,cartilage, fat, epithelium, and such.

In addition, the colonies obtained as a result of culturing, on a solidphase, peripheral blood after administration of a peptide consisting ofamino acid residues 1-44 of the human HMGB1 protein (hereinafter, HMGB1peptide 1-44), which has an activity of mobilizing mesenchymal stemcells into peripheral blood, have been confirmed to have adherability tosolid phase and self-renewal ability, and to be PDGFRα positive. Theyhave also been confirmed to have a gene expression profilecharacteristic to mesenchymal stem cells, based on the results ofclustering the transcriptome analysis data and performing gene ontologyanalysis.

Furthermore, it has been confirmed that the number of colonies obtainedby solid-phase culture is larger in the peripheral blood afteradministration of HMGB1 peptide 1-44 than in the peripheral blood afteradministration of physiological saline.

Therefore, the colonies obtained as a result of culturing peripheralblood on a solid phase are mesenchymal stem cells, and it is consideredthat an increase in the number of colonies detected in a solid phaseculture of peripheral blood indicates an increase in the number ofmesenchymal stem cells in the peripheral blood.

In addition, since usually mesenchymal stem cells are rarely present inperipheral blood, it is thought that the increased amount of mesenchymalstem cells was mobilized into peripheral blood from tissues other thanperipheral blood (for example, bone marrow).

From the above, the number of colonies detected in the solid-phaseculture of peripheral blood after administration of a test substance canbe used as an indicator of the activity of the test substance tomobilize mesenchymal stem cells into peripheral blood.

(2) Result

In the mice administered with any of the peptides NP-1 to NP-11, thenumber of colonies obtained on the plate by culturing the peripheralblood-derived cells was larger than that in the mice administered withphysiological saline (FIGS. 1 to 4).

As described above, an increase in the number of colonies detected bythe colony assay described herein indicates an increase in the number ofmesenchymal stem cells in peripheral blood, and thus these resultsdemonstrate that the fragment peptides of the nuclear proteins have theactivity of mobilizing mesenchymal stem cells into peripheral blood.

Example 2 Efficacy of Nuclear Protein-Derived Peptides for InflammatoryBowel Disease (1) Materials and Methods i) Drugs

Dextran sulfate sodium (DSS) (molecular weight 5,000-6,000, manufacturedby Nacalai Tesque Inc., Catalog No. 10930-94) was dissolved in water toprepare a 2.5% (w/v) DSS aqueous solution. In addition, the peptidesNP-1 to NP-4 (all TFA salts) described in Example 1 were used as testsubstances.

ii) Generation of the Inflammatory Bowel Disease (IBD) Model Mice

C57BL/6J mice (8 weeks old, male, body weight about 20 g) were allowedfree drinking of 2.5% DSS aqueous solution in place of purified water(RO water) to induce colitis (drinking of the DSS aqueous solution wascontinued for 10 days).

iii) Peptide Administration

The IBD model mice prepared as described above were divided into peptideadministration groups (NP-1 to NP-4, each n=3) and a control group(n=3). Administration of each peptide was performed on Days 1, 3, 5 and7 after the start of drinking the DSS aqueous solution, by adjusting thepeptide solution to a concentration of 0.5 mg/mL using physiologicalsaline as the solvent, and injecting the amount of 200 μL/animal (5mg/kg dose of peptide) into the tail vein. In the control group, 200μL/animal of physiological saline was injected into the tail vein onDays 1, 3, 5 and 7 after the start of drinking the DSS aqueous solution.

iv) Evaluation of the Effect of Peptide Administration

Mice were weighed daily for 10 days from the start of drinking the DSSaqueous solution.

(2) Result

Changes in the body weight of mice during the test period are shown inFIGS. 5 to 8 (see “Saline” for the control group and “NP-1”, “NP-2”,“NP-3”, “NP-4” for the peptide NP-1 to NP-4 administration groups.). Thebody weight of the control group decreased with the passage of days, andwas about 87% on the 10^(th) day after the start of drinking the DSSaqueous solution as compared with that before the start of the DSSdrinking. On the other hand, in all of the peptide NP-1 to NP-4administration groups, the body weight loss was suppressed as comparedwith the control group, and the body weight on the 10^(th) day after thestart of drinking the DSS aqueous solution was significantly larger thanthat in the control group.

One of the symptoms of IBD is known to be occurrence of body weightloss, and the reason is thought to be malnutrition due to inflammationand tissue damage (such as ulcer) that occur in the intestinal mucosa.Furthermore, intravenous injection of mesenchymal stem cells in ananimal IBD model is known to improve various symptoms including bodyweight loss, epithelial damage in the intestinal tract, infiltration ofinflammatory cells, and such. Such improvement of symptoms is due tosuppression of inflammation of the intestinal mucosa by theanti-inflammatory effect of mesenchymal stem cells, and resultingfacilitation of mucosal tissue regeneration, and such.

This time, by administering fragment peptides of nuclear proteins of thepresent application to the IBD model mice, the body weight loss wassuppressed. This is thought to be the result of mobilization ofmesenchymal stem cells into peripheral blood by the action of thefragment peptides of the nuclear proteins, and exhibition of theinflammation-suppressing effect and tissue regeneration effect by thecells.

Example 3 Efficacy of Nuclear Protein-Derived Peptides on Psoriasis (1)Materials and Methods i) Drugs

To induce psoriasis by imiquimod, a cream containing 5% imiquimod(Beseruna cream 5%, Mochida Pharmaceutical Co., Ltd.) was used. In thedrawing corresponding to the Example of the present application,imiquimod is referred to as IMQ. In addition, the peptide NP-3 (TFAsalt) described in Example 1 was used as a test substance.

ii) Generation of Psoriasis Model Mice

C57BL/6 mice (7 weeks old, female, body weight about 20 g) wereprepared. To induce psoriasis, a cream containing 5% imiquimod wasapplied on the auricular skin of the mice at an amount of 25 mg/ear/day(1.25 mg/ear/day imiquimod) once a day for seven days. Moreover, micewithout imiquimod application (hereinafter, referred to as “normalmice”) were used as a comparison target.

iii) Peptide Administration

The psoriasis model mice prepared as described above were divided into apeptide-administered group (n=4) and a control group (n=4).Administration of the test substance was carried out from the first dayof the start of imiquimod application (Day 1) for seven days, byinjecting into the tail vein 100 μL/day of the NP-3 peptide solutionwhich has been adjusted to a concentration of 1 using physiologicalsaline as a solvent (5 mg/kg/day as peptide dose). In the control group,physiological saline was injected into the tail vein in an amount of 100μL/day, from the first day of the start of imiquimod application forseven days. No substance was administered to normal mice (n=4).

iv) Evaluation of the Effects of Peptide Administration

During the test period, the auricle thickness of mice was measured dailyusing a micrometer (manufactured by Mitutoyo Co., Ltd., Product No.CLM1-15QM), and the size of change from the auricle thickness before thestart of imiquimod application (Day 0) was calculated. The degree ofskin thickening was evaluated using the size of change as an index.

(2) Result

Changes in auricular thickness of mice during the test period are shownin FIG. 9 (see “Non treat” for normal mice, “IMQ/saline” for the controlgroup, and “IMQ/NP-3” for the peptide NP-3 administration group). Theauricular thickness of psoriasis model mice (the control group andpeptide NP-3 administration group) increased with the passage of days.In the peptide NP-3 administration group, the increase in auricularthickness was suppressed as compared with the control group.

Symptoms of psoriasis vulgaris include erythema, thickening,scales/desquamation of the skin, which are caused by abnormalities inthe immune system and the resulting inflammation, and hyperproliferationof keratinocytes. The mouse model used in this experiment is a modelthat causes psoriasis vulgaris-like symptoms (erythema and thickening)by applying imiquimod to the skin of the ear. As a result of theexperiment, imiquimod-induced skin thickening was suppressed byadministration of fragment peptide of the nuclear protein of the presentapplication. This is thought to be the result of mobilization ofmesenchymal stem cells into peripheral blood by the effect of thefragment peptide of the nuclear protein, and exhibition of the immunemodulatory effect and inflammation-suppressing effect by the cells.

INDUSTRIAL APPLICABILITY

The nuclear proteins or fragment peptides thereof of the presentapplication can be used as therapeutic agents for inflammatory diseases,autoimmune diseases, fibrotic diseases, and diseases accompanied bytissue damage/ischemia/necrosis.

1. A method for mobilizing mesenchymal stem cells to peripheral blood,comprising administering to a subject an effective amount of a nuclearprotein or a fragment peptide thereof.
 2. A method for treating adisease or pathological condition in a subject by mobilizing mesenchymalstem cells to peripheral blood, which comprises administering to thesubject an effective amount of a nuclear protein or a fragment peptidethereof.
 3. The method of claim 2, wherein the treatment of a disease orpathological condition is selected from inflammation-suppressingtherapy, immunomodulatory therapy, tissue regeneration-inducing therapy,and tissue fibrosis-suppressing therapy.
 4. The method of claim 2,wherein the disease or pathological condition is selected from aninflammatory disease, an autoimmune disease, a disease accompanied bytissue damage, ischemia, or necrosis, and a fibrotic disease.
 5. Themethod of claim 2, wherein the disease or pathological condition isselected from inflammatory bowel disease and psoriasis.
 6. A method fortreating a disease selected from inflammatory bowel disease or psoriasisin a subject, which comprises administering to the subject an effectiveamount of a nuclear protein or a fragment peptide thereof.
 7. The methodof claim 2, wherein the nuclear protein or fragment peptide thereofcomprises a nuclear localization signal.
 8. The method of claim 2,wherein the nuclear protein or fragment peptide thereof is a nuclearprotein involved in transcription regulation or a fragment peptidethereof.
 9. The method of claim 2, wherein the nuclear protein orfragment peptide thereof is a nuclear protein selected from thefollowing or a fragment peptide thereof: (1) BTF3 protein; (2) SUPT16Hprotein; (3) YBX1 protein; (4) NPM1 protein; (5) PA2G4 protein; (6)PFDN5 protein; (7) PSMC3 protein; (8) HNRNPK protein; and (9) a nuclearprotein functionally equivalent to a protein selected from (1) to (8).10. The method of claim 2, wherein the nuclear protein or fragmentpeptide thereof is a nuclear protein selected from the following or afragment peptide thereof: (a) a nuclear protein comprising an amino acidsequence selected from SEQ ID NOs: 1 to 34; (b) a nuclear proteincomprising an amino acid sequence resulting from substitution, deletion,insertion, or addition of one or more amino acids in an amino acidsequence selected from SEQ ID NOs: 1 to 34; and (c) a nuclear proteincomprising an amino acid sequence with a sequence identity of about 80%or higher with an amino acid sequence selected from SEQ ID NOs: 1 to 34.11. The method of claim 2, wherein the fragment peptide of the nuclearprotein is a fragment peptide selected from the following: (a) a nuclearprotein fragment peptide consisting of a portion of an amino acidsequence selected from SEQ ID NOs: 1 to 34; (b) a nuclear proteinfragment peptide comprising an amino acid sequence selected from SEQ IDNOs: 35 to 56; (c) a nuclear protein fragment peptide consisting of aportion of an amino acid sequence selected from SEQ ID NOs: 35 to 56;(d) a nuclear protein fragment peptide comprising an amino acid sequenceresulting from substitution, deletion, insertion, or addition of one ormore amino acids in an amino acid sequence selected from SEQ ID NOs: 35to 56; and (e) a nuclear protein fragment peptide comprising an aminoacid sequence with a sequence identity of about 80% or higher with anamino acid sequence selected from SEQ ID NOs: 35 to
 56. 12. A fragmentpeptide of a nuclear protein selected from the following: (1) BTF3protein; (2) SUPT16H protein; (3) YBX1 protein; (4) NPM1 protein; (5)PA2G4 protein; (6) PFDN5 protein; (7) PSMC3 protein; (8) HNRNPK protein;and (9) a nuclear protein functionally equivalent to a protein selectedfrom (1) to (8).
 13. The fragment peptide of claim 12, comprising anuclear localization signal.
 14. The fragment peptide of claim 12, whichis a fragment peptide of a nuclear protein selected from the following:(a) a nuclear protein comprising an amino acid sequence selected fromSEQ ID NOs: 1 to 34; (b) a nuclear protein comprising an amino acidsequence resulting from substitution, deletion, insertion, or additionof one or more amino acids in an amino acid sequence selected from SEQID NOs: 1 to 34; and (c) a nuclear protein comprising an amino acidsequence with a sequence identity of about 80% or higher with an aminoacid sequence selected from SEQ ID NOs: 1 to
 34. 15. The fragmentpeptide of which is selected from the following: (a) a nuclear proteinfragment peptide consisting of a portion of an amino acid sequenceselected from SEQ ID NOs: 1 to 34; (b) a nuclear protein fragmentpeptide comprising an amino acid sequence selected from SEQ ID NOs: 35to 56; (c) a nuclear protein fragment peptide consisting of a portion ofan amino acid sequence selected from SEQ ID NOs: 35 to 56; (d) a nuclearprotein fragment peptide comprising an amino acid sequence resultingfrom substitution, deletion, insertion, or addition of one or more aminoacids in an amino acid sequence selected from SEQ ID NOs: 35 to 56; and(e) a nuclear protein fragment peptide comprising an amino acid sequencewith a sequence identity of about 80% or higher with an amino acidsequence selected from SEQ ID NOs: 35 to 56.